Microbial dysbiosis and fecal metabolomic perturbations in Yorkshire Terriers with chronic enteropathy

Dysbiosis and perturbations of fecal metabolic profiles have been reported in dogs with inflammatory bowel disease. Currently the incidence of dysbiosis and the fecal metabolomic profile in Yorkshire Terriers with chronic enteropathy (YTE) and the effects of treatment are unknown. This prospective observational study analyzed the dysbiosis index (DI) and fecal bile acid, sterol and fatty acid profiles in 14 Yorkshire Terriers with active YTE, 11 dogs in clinical remission, and 26 healthy Yorkshire Terriers. YTE was associated with dysbiosis and a significant increase in fatty acids (docosanoate, p = 0.002; gondoate, p = 0.026; erucate, p < 0.001; nervonate, p < 0.001; linolenate, p < 0.001), and plant sterols (campesterol, p < 0.001; brassicasterol, p = 0.024). The abundances of Fusobacterium (p < 0.001) and Cl. hiranonis (p = 0.018) and the concentrations of the secondary bile acid ursodeoxycholic acid (p = 0.033) and the plant sterol sitostanol (p = 0.003) were significantly decreased compared to healthy dogs. Dysbiosis, abundances of Fusobacterium, Cl. hiranonis and fecal concentrations of bile acids and sterols did not recover after treatment, while fecal fatty acid concentrations decreased in treated dogs. YTE is associated with dysbiosis and changes in bile acid, fatty acid, and sterol metabolism. These changes only recovered partially despite clinical remission. They might be breed-specific and involved in the pathogenesis of YTE.

Endoscopic duodenal scores ranged from 0 to 3 with a median score of 2 (maximal possible score 4). Histological duodenal World Small Animal Veterinary Association (WSAVA) scores indicated mild or moderate histological changes (median 4.5, range [2][3][4][5][6][7][8][9][10][11]. In all dogs a predominantly lymphoplasmacytic infiltration of the intestinal mucosa was found. Seven dogs showed an additional eosinophilic and four dogs an additional neutrophilic infiltration. Other histological abnormalities were villus blunting in 4/11 dogs where villus length could be evaluated, increased intraepithelial lymphocytes (3/14), and fibrosis of the propria (3/14). Furthermore, crypt lesions and lymphangiectasia were present in 4 and 3 dogs, respectively. YTE in clinical remission. Eleven dogs were available for re-evaluation after achieving clinical remission. Nine dogs were clinically well controlled receiving a hydrolyzed diet. In 8 of them the hydrolyzed diet was the first diet that was introduced. One dog was switched from a low fat to a hydrolyzed diet before achieving clinical remission. Fecal sampling of the dogs was repeated 69-119 days (median 70 days) post diagnosis. One dog was clinically well controlled receiving a low fat diet and was sampled after 70 days. One dog achieved clinical remission after the administration of prednisolone and was sampled again 126 days post diagnosis (71 days after initiation of prednisolone). One dog did not finish the study due to poor owner compliance. Two dogs had not finished the treatment trial at the time of sample analysis. The CCECAI scores had decreased in all re-evaluated dogs (n = 11, median 2, range 1-3).
Healthy control group. The control group (n = 26) consisted of 14 female (8 spayed) and 12 (6 neutered) male Yorkshire Terriers. The mean age of the dogs was 8.2 years (+/−3.2 years). The mean body weight was 3.9 kg (+/−1.35). Twenty-five dogs in the control group received various commercial diets (19 canned and 6 dry foods). One dog was fed with a Bones and Raw Food (BARF) diet. Sex and body weight did not differ significantly between healthy and YTE dogs. Dogs in the healthy control group were older than dogs with YTE (p = 0.04). The median CECCAI score in the healthy control group was 0.5 (range 0-3), which was significantly lower compared to dogs with YTE (p < 0.001).

Dysbiosis index and fecal metabolomics in YTE dogs compared to healthy control dogs. Dys-
biosis Index. Seven YTE dogs had DI values > 2, indicating a shift in the intestinal microbiome. In 1 dog the DI was mildly increased (0-2), suggesting a minor shift in the microbiota. Six YTE dogs had a negative DI, but in 5 of these dogs the abundance of individual bacteria was outside the respective reference interval, indicating minors forms of dysbiosis in those dogs as well. In all of these 5 dogs the abundance of Faecalibacterium was decreased, either as a single change or in combination with other altered groups. The abundance of Clostridium hiranonis (Cl. hiranonis) was decreased in 7 dogs with YTE and that of Faecalibacterium was decreased in 8 dogs with YTE. Of the healthy control dogs 4 had a DI > 2 and 4 had values between 0 and 2, suggesting that several of the control dogs also had shifts in the intestinal microbiome. Eighteen healthy control dogs had negative DI values, with 3 of them having an abundance of one bacterial group outside the respective reference interval, indicating a minor form of dysbiosis.
The DI was significantly higher in dogs with YTE compared to healthy control dogs (p = 0.048). The abundances of Fusobacterium (p < 0.001) and Cl. hiranonis (p = 0.018) were significantly lower in dogs with YTE compared to healthy control dogs (Table 1; Fig. 1). The DI correlated positively with CCECAI scores in YTE dogs (ρ = 0.55, p = 0.042).
Fecal bile acid concentrations. The concentration of ursodeoxycholic acid (UDCA), a secondary bile acid, was significantly lower in the feces of YTE dogs compared to healthy control dogs (p = 0.033) ( Table 2; Fig. 2).
In YTE dogs, total fecal primary bile acid concentrations and the percentage of primary bile acids correlated negatively with the abundance of Cl. hiranonis (ρ = − 0.70, p = 0.006; ρ = − 0.67, p = 0.009) and positively with the DI (ρ = 0.70, p = 0.006; ρ = 0.63, p = 0.016). Concentrations and percentages of total secondary bile acids correlated negatively with the DI (ρ = − 0.60, p = 0.022; ρ = − 0.63, p = 0.016). Percentages of fecal secondary bile acids Dysbiosis Index and fecal metabolomics in active YTE compared to YTE dogs in remission. Dysbiosis Index. In none of the YTE dogs with DI values >0 at the time of enrolment, the DI recovered after achieving clinical remission. In only 1 dog with decreased abundance of Faecalibacterium and a negative DI, Faecalibacterium abundance reached the reference interval after treatment. In none of the dogs the abundance of Cl. hiranonis recovered after achieving clinical remission. There was no statistically significant difference of the DI or the abundances of Cl. hiranonis and Fusobacterium between YTE dogs before and after treatment. (Table 5; Fig. 1).
Fecal bile acid concentrations. The concentrations and the percentages of total fecal secondary bile acids as well as the ratio of secondary to primary bile acids decreased significantly in dogs with YTE after treatment. The concentrations and percentages of the secondary fecal bile acids lithocholic acid (LCA) and deoxycholic acid (DCA), as well as the percentage of the secondary bile acid chenodeoxycholic acid (CDCA) were significantly decreased in YTE dogs in clinical remission. Conversely, the percentage of total fecal primary bile acids and the Table 2. Summary statistics of fecal bile acid concentrations in healthy and YTE dogs. CA, cholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; TPBA, total primary bile acids; TSBA, total secondary bile acids; SBA to PBA; ratio of secondary to primary bile acids; PBA to SBA, ratio of primary to secondary bile acids; YTE, Yorkshire Terrier enteropathy; SD, standard deviation.  www.nature.com/scientificreports/  www.nature.com/scientificreports/ percentage of the primary bile acid cholic acid (CA) increased significantly in YTE dogs in remission. (Table 6; Fig. 2).
Fecal sterol concentrations. There was no statistically significant difference in any of the measured fecal sterol concentrations of the dogs during active disease or during clinical remission. (Table 7; Fig. 3).

Discussion
In the current study, the DI was used as a quantitative tool for assessment of intestinal dysbiosis and quantitative targeted metabolomics were used to identify changes in fecal metabolites in dogs with YTE compared to healthy Yorkshire Terriers, and to investigate the impact of treatment on both the microbiome and the fecal metabolome. This study revealed a high incidence of dysbiosis, an increased DI, and a lower abundance of Fusobacterium and Cl. hiranonis in dogs with YTE, compared to healthy Yorkshire Terriers. Furthermore, we identified changes in lipid and bile acid metabolism in symptomatic dogs with YTE. However, these changes were only partially resolved in dogs in clinical remission.
In accordance to previous studies in dogs with CE 10,17,18 , dogs with YTE had a higher DI compared to healthy control dogs, indicating gut dysbiosis. Furthermore, the DI increased with increased clinical severity of YTE. Gut dysbiosis refers to a disturbance of the intestinal microbiome, leading to functional deviations of the microbial transcriptome, proteome, or metabolome 7 . The DI is based on a mathematical algorithm, which was developed based on the results of molecular studies 19,20 , to calculate the degree of dysbiosis in dogs using fecal samples 8 . The fecal DI uses the quantification of the abundance of total bacteria and 7 bacterial groups (Faecalibacterium spp., Turicibacter spp., Escherichia coli, Streptococcus spp., Blautia spp., Fusobacterium spp., and Cl. hiranonis) that are commonly altered in dogs with CE. Based on the quantitative measurements by qPCR, reference intervals have been established for the fecal abundance of each bacterial group. A negative DI indicates that no shifts in the overall diversity of the intestinal microbiota have occurred. However, the abundance of individual bacterial groups outside their reference intervals suggests mild dysbiosis. A DI between 0 and 2 is currently defined as a mild to moderate shift in the intestinal microbiota and values > 2 are indicative of major shifts in the intestinal    10,17,18 Cl. hiranonis has been shown to play an important role in the conversion of primary bile acids to secondary bile acids 22 and decreases in the fecal abundance of Cl. hiranonis have been reported in dogs with CE 8,12 . Fusobacterium is associated with the production of short chain fatty acids from protein sources 23 . Decreased fecal abundances of Faecalibacterium, a short chain fatty acid producer, have also been reported in dogs with CE 10,13,17 . All three bacterial groups play a crucial role in lipid and bile acid metabolism and have a major influence on the gut metabolome. Metabolomic profiling allows for the analysis of small molecules in biological samples. It is a powerful tool to analyze metabolic changes associated with several pathological conditions. It is increasingly used for the discovery of etiological factors, disease signatures, and as a screening tool for different pathological conditions, including IBD 24 . Characterization of metabolic changes occurring in canine CE can increase our understanding of disease pathophysiology, which may improve diagnosis, treatment, and disease management. In this study, dogs with YTE had significantly lower fecal concentrations of UDCA, a secondary bile acid, compared to healthy controls. This is consistent with recent studies revealing decreased secondary bile acid concentrations in fecal samples of dogs with CE 11,13,17 . A substantial proportion of humans with Crohn's disease Table 6. Summary statistics of bile acid concentrations in YTE dogs before and after treatment. CA, cholic acid; CDA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; TPBA, total primary bile acids; TSBA, total secondary bile acids; SBA to PBA; ratio of secondary to primary bile acids; PBA to SBA, ratio of primary to secondary bile acids; YTE, Yorkshire Terrier enteropathy; SD, standard deviation. www.nature.com/scientificreports/ and with diarrhea-predominant irritable bowel syndrome suffer from a condition known as bile acid or bile salt malabsorption, which is characterized by elevated total and primary fecal bile acids 25 . Furthermore, decreased expression of the active sodium-dependent bile acid transporter in the ileum of dogs with chronic enteropathy has been documented, indicating bile acid malabsorption in dogs with CE 18 . Interestingly, in our study the concentrations of total fecal bile acids correlated positively with increased severity of histological lesions in dogs with YTE. However, it seems that a disbalance rather than an excessive amount of colonic bile acids is more common in canine CE. Primary bile acids are synthesized by the liver and secreted into the intestine. In healthy animals, a small percentage of the intestinal bile acids are not reabsorbed and reach the colon, where colonic bacteria convert primary to secondary bile acids by deconjugation and dehydroxylation 26 . In our study we found a negative correlation of fecal primary bile acids with Cl. hiranonis abundance, while secondary bile acids were positively correlated with the abundance of this specific bacterial group. This supports the role of Cl. hiranonis as an important bile acid converter in dogs. YTE and dysbiosis may lead to bile acid dysmetabolism due to a decreased proportion of Cl. hiranonis, resulting in altered bile acid conversion. The current study revealed perturbations in lipid metabolism in dogs with YTE. Lipids are a heterogeneous group of hydrophobic and amphiphilic molecules that are responsible for the maintenance of membrane structure and permeability and possess many regulatory functions in immunity and inflammation 27 . In this study, YTE dogs had increased fecal concentrations of the plant sterols campesterol and brassicasterol, while the plant sterol metabolite sitostanol concentrations were decreased. Decreased sitostanol concentrations have been previously described in dogs with IBD 28 . Sterols are a subtype of lipids occurring in cell membranes. While cholesterol is an essential component of animal cell membranes, sterols are the structural components of plant cell membranes. The intestinal absorption of cholesterol is significantly more efficient (30-60%) than plant sterol absorption (2-3%). However, plant sterols can reduce cholesterol absorption in the gastrointestinal tract and therefore have been studied mostly in relation to their hypocholesteremic effects 29 . Since plant sterols cannot be synthesized de novo by mammalian hosts, fecal concentrations resemble the end result of dietary ingestion and intestinal Table 7. Summary statistics of fecal sterol concentrations in YTE dogs before and after treatment. TM, total measured; TM Phyto-to Zoosterols; ratio of total measured phyto-to total measured zoosterols; TM Sterols to Fatty Acids; ratio of total measured sterols to total measured fatty acids; YTE, Yorkshire Terrier enteropathy; SD, standard deviation. www.nature.com/scientificreports/ absorption. Therefore, malabsorption due to disturbed epithelial transport, a reduced intestinal absorptive area, and/or accelerated intestinal transit time accompanying gastrointestinal disease could all be reasons for increased fecal concentrations of plant sterols 30,31 . However, this would not explain decreased sitostanol concentrations identified in our study. Sterols do not pass the gastrointestinal tract unchanged, but unabsorbed sterols reach the colon where they are metabolized by intestinal bacteria 29,32 . Therefore, dysbiosis as seen in YTE dogs of our study could lead to disbalances in fecal sterols and their metabolites. We identified a positive correlation of fecal sitostanol concentrations and the fecal abundance of Faecalibacterium. The microbes responsible for sterol metabolism in dogs are unknown and the fecal abundance of Faecalibacterium was below the lower limit of the reference interval in the majority of dogs with YTE. This could indicate a role for Faecalibacterium in plant sterol metabolism or, alternatively, an influence of sterols on the abundance of Faecalibacterium. Altered fecal sterol concentrations could serve as markers for dysbiosis, and given the anti-inflammatory role of plant sterols in IBD animal models [33][34][35] , they could also play a role in the chronic inflammatory loop of YTE. The current study revealed a significant increase of fecal long-chain free fatty acids in dogs with YTE, indicating lipid malabsorption or dysmetabolism. Elevated fecal fatty acid concentrations have been reported in human IBD patients, which is consistent with our results 36 . Increased fecal fatty acids could be a direct measure of fatty acid loss as a consequence of intestinal malabsorption. Decreased intestinal fatty acid absorptive capacity may be caused by disturbed epithelial transport, reduced intestinal absorptive area and/or accelerated intestinal transit time 30,31 . Moreover, YTE is often associated with dysfunctional intestinal lymphatics 14,15 , which may cause breed specific alterations in lipid absorption. Alternatively, altered fecal fatty acid profiles may indicate altered lipid cascades in inflammation and immune regulation. Fatty acids are known to play important roles in inflammation and immunity, trafficking, signal transduction, regulation of gene expression and autophagy. Furthermore, they are involved in the maintenance of the intestinal barrier and serve as antioxidants 27 . It has been shown that colonic inflammation in human IBD patients and IBD mouse models is associated with altered intestinal mucosal lipid profiles, which appear to be dependent on the degree of inflammation 37 . Similarly, we found that the concentration of individual fecal fatty acids increased with increased histological severity of intestinal lesions in YTE dogs. Fecal fatty acid concentrations could serve as markers for intestinal dysfunction or inflammation. Furthermore, they could be involved in a detrimental feedback loop where intestinal disease results in reduced fatty acid absorption and increased colonic long chain fatty acids may lead to diarrhea and epithelial cell damage 38-40 . Normalization of DI or metabolomic profiles in dogs in clinical remission would be useful to monitor therapeutic responses in dogs with YTE and to identify patients with a disease relapse before the onset of clinical www.nature.com/scientificreports/ signs to allow early intervention. However, in the current study, clinical recovery of dogs with YTE did not correlate with the recovery of dysbiosis. A recent study demonstrated that although the DI in dogs with steroidresponsive CE normalized after three weeks of treatment, several bacterial taxa evaluated by qPCR and 16S rRNA sequencing were significantly different compared with healthy controls and did not normalize until one year after treatment response 41 . Another study in dogs with food responsive CE reported similar results with no recovery of the intestinal microbiome analysed by qPCR and 16S rRNA sequencing after a treatment period of 60 days 42 . Furthermore, a previous study evaluating the recovery of the DI in dogs with steroid responsive CE revealed that the DI was still increased after 3 months, despite clinical remission 17 . These data suggest that it takes several months to years for the gut microbiome to recover, likely due to changes at the intestinal mucosal level, and the timeframe chosen for reassessment in our study may have been insufficient to reach normobiosis. In addition, in treated YTE dogs of our study, bile acid dysbalance became even more pronounced with a decrease of various individual and total secondary bile acids and an increase in fecal primary bile acid concentrations. This is in contrast to results of a previous study in dogs with steroid responsive CE in which the fecal abundance of Cl. hiranonis significantly increased and bile acid metabolism was restored after two months of treatment 17 . Corticosteroids may improve bile acid metabolism by upregulation of the ileal active sodium-dependent bile acid transporter 43 , however this would not explain the recovery of Cl. hiranonis abundance. Furthermore, similar results were seen in dogs with CE reaching clinical remission after receiving a hydrolyzed protein diet 44 . Contrary to our results, the abundance of Cl. hiranonis increased and fecal bile acid concentrations normalized in dogs with food responsive disease after 2 weeks; which was in contrast to non-responders 44 . Therefore, it was suggested by the authors, that remission induced by a hydrolyzed protein diet is associated with improved dysbiosis, expansion of bile acid converters and, increasing concentrations of fecal secondary bile acids 44 . At least this does not appear to be the case in the dogs with YTE in our study, in which clinical recovery did not correlate with recovery of Cl. hiranonis abundance and bile acid dysbalance. In accordance to our results, feeding an extruded animal protein free diet to dogs with CE and healthy controls did not have an influence on the abundance of Cl. hiranonis 42 and feeding a hydrolyzed diet in healthy dogs did not have an impact on Cl. hiranonis abundance or fecal bile acid concentrations 45 . While it is believed that dysbiosis represents the sequel rather than the cause of CE, we cannot exclude that gut dysbiosis and dysregulated intestinal metabolism is a predisposing factor for YTE and might persist in these dogs.
Similarly to the fecal DI and bile acid concentrations, despite clinical improvement fecal sterol concentrations did not normalize in YTE dogs. This might be expected if dysbiosis is responsible for an altered sterol pattern.
In contrast, long-chain fatty acids were recovered from the feces of dogs in clinical remission. This might indicate improved absorption by recovered lymphatics and intestinal mucosa and/or decreased intestinal inflammation. However, we did not reassess small intestinal biopsies in the treated IBD dogs of the present study, so we can only speculate on the intestinal histopathological appearance after clinical remission. Previous studies have shown that the histopathologic lesions in intestinal biopsies of dogs suffering from IBD did not change despite clinical improvement [46][47][48][49] . On the other hand, in a recent study feeding an omega-3 enriched diet increased the treatment response and resulted in marked suppression of intestinal inflammatory activity in IBD dogs 50 . Another explanation for our results could be that the diet itself had a beneficial influence on long-chain fatty acids by higher digestibility or a different fatty acid pattern.
Our study is not without limitations. The main limitation is the small number of dogs included, especially in the remission group. Another limitation is that dogs in the control group were older than dogs with YTE, which might have influenced our results. A previous study revealed that the canine intestinal gut microbiome showed a decreased microbial diversity and is likely to vary with increasing age 51 . However, the influence of age on the DI and the fecal metabolome in adult dogs have not yet been studied. Due to ethical concerns, we did not perform gastroduodenoscopy in dogs in clinical remission to assess recovery of intestinal inflammation and architectural changes. Furthermore, since we enrolled client-owned dogs, the dogs were not fed a standardized diet, which might have influenced the results of metabolite profiling. However, at the time of enrolment all dogs except one (receiving a BARF diet) were fed comparable commercially available diets. Additionally, in one dog in the treatment group prednisolone had to be added to the diet. This may have influenced our results, since prednisolone may have a direct effect on individual metabolites, and dietary non responders may represent a more severe disease phenotype. Other conditions causing intestinal inflammation or epithelial injury may share the observed alterations. To ensure clinical relevance, it might be necessary to include Yorkshire Terriers with other gastrointestinal disorders in the future.
In summary, our study showed changes in the fecal microbiome and the fecal metabolic profile in Yorkshire Terriers with YTE. Notably, a dysbalance in bile acid metabolism, sterols, and fatty acids was observed in YTE patients. Considering the important functions of these compounds in inflammation, further studies are needed to shed more light on the role of these metabolites in dogs with YTE. Recognizing and understanding breed specific differences provides a great opportunity to optimize our treatment approach in dogs with IBD. www.nature.com/scientificreports/ 2018 and March 2021. The diagnosis of YTE was based on the results of physical examination, haematological and biochemical parameters, urine analysis, abdominal ultrasonography, fecal parasitological examinations and gastroduodenoscopy results. For the exclusion of systemic, infectious, endocrine, and neoplastic causes for the presenting signs a complete blood count, serum biochemical analysis, the measurement of serum total bile acids, basal cortisol concentrations, cTLI (canine trypsin-like-immunoreactivity) and cPLI (canine pancreatic lipase immunoreactivity as measured by Spec cPL) and an ACTH-stimulation test (if the basal cortisol < 2 µg/dl) were performed. Additionally analysis of fecal samples by flotation and Giardia antigen test and abdominal ultrasonography were performed. Furthermore, for the exclusion of renal protein loss urinalysis including evaluation of the urine sediment and urine protein creatinine ratio (UPC) was performed. To confirm intestinal mucosal inflammation all YTE cases underwent gastroduodenoscopy. The control group consisted of 26 adult Yorkshire Terriers without signs of gastrointestinal disease (n = 26). The dogs were prospectively enrolled in the study over the same time period and were healthy as determined by history, physical examination, blood count, chemical profile, fecal and urinalysis, and abdominal ultrasonography. Dogs with antibiotic or glucocorticoid pretreatment within the last 2 weeks or dogs that had received a specific diet designed for dogs with gastrointestinal disease within the last 2 months were excluded from the study.

Materials and methods
Clinical scores were collected from all dogs using the canine chronic enteropathy activity index (CCECAI) 46 at the time of study entry and additionally for YTE dogs at the control appointments. The initial diagnostic workup was performed by one board-certified internist (A.I.G.), who also performed the control visits. Endoscopic duodenal activity scores were calculated based on the presence (1 point each) or absence (0 points each) of friability, granularity, erosions, and lymphatic dilatation 52 .
Intestinal biopsies of the YTE dogs were graded by one blinded board-certified pathologist (B.R.), according to the World Small Animal Veterinary Association (WSAVA) International Gastrointestinal Standardization Group guidelines 2 .
All dogs with YTE were part of a treatment trial consisting of the initial feeding of either a hydrolyzed diet (Hill's Prescription Diet z/d Canine) or a low fat diet (Hill's Prescription Diet i/d Low Fat Canine). The diet was randomly assigned by random number generation and the owners and the specialist performing the work up and control appointments were blinded to the diets. The dogs were re-evaluated every 14 days. If the treatment was not successful (CCECAI score > 3), the dogs were switched to the other diet and rechecked after another 14 days. A weaning period was not considered for ethical reasons. The feeding of canned or dry food was at the discretion of the owner to increase the acceptability of the diet. The amount of feed was according to the manufacturer's instructions based on body weight. In case of non-response to dietary treatment alone (CCECAI > 3), additional administration of prednisolone (1 mg/kg q12h PO) was planned. In case of further non-response after another 14 days, cyclosporine (5 mg/kg q24h PO) would have been added as a second immunosuppressant. After achieving clinical remission (defined by a decrease in CCECAI scores to ≤ 3), at the earliest 70 days after treatment initiation, YTE dogs were re-examined, and fecal samples were again collected for DI and metabolomic analysis. Fecal Dysbiosis Index. For the calculation of the DI, quantitative PCR assays were performed for total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, and Clostridium hiranonis as previously described 8,13 . The results were expressed as log DNA abundance (femtogram) for each particular bacterial group per 10 nanogram of total isolated DNA.
Fecal metabolome analysis. A targeted metabolomic approach was used to measure fecal concentrations of selected unconjugated bile acids (i.e., cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, ursodeoxycholic acid), cholesterol and cholesterol intermediates (i.e., coprostanol, cholestanol, lathosterol), plant sterols (i.e., β-sitosterol, brassicasterol, campesterol, fucosterol, sitostanol, stigmasterol) and long chain fatty acids (i.e., palmitate, linoleate, α-linolenate, oleate, cis-vaccenate, stearate, arachidonate, gondoate, erucate, docosonoate, nervonate). A previously described protocol for the simultaneous quantitation of fatty acids, sterols and bile acids in human stool was adapted and modified 53 . An aliquot of 10-14 mg of a lyophilized fecal sample was weighted in a 7 mL glass centrifuge tube for further sample processing. The weight of each sample was recorded for the calculation of the final concentration. A mixture of 160 μL 1-butanol, 10 μL each d7-sitostanol, d6-cholesterol, d4-stearic acid, and d4-cholestane (2 mg/mL each), and 20 μL each of d4-cholic acid and d4-lithocholic acid (1 mg/mL each) was added to each sample. A 20 μL concentrated HCl was added to each tube and vortexed for at least 30 seconds, before the tube was incubated at 65 °C for 4 hours (ThermoScientific, REACTI-Therm III #TS-18824 Heating module). After 4-hour incubation, samples were briefly vortexed, then dried with heat on under nitrogen gas until visibly dry. A 200 μL of a commercial silylating mixture (Sylon HTP, a mixture of hexamethyldisilazane, chlorotrimethylsilane, and pyridine = 2:1:10 (v/v/v)) was added to each dried tube for 30-min incubation at 65 °C. After incubation, tubes were again vortexed briefly before drying under heat and nitrogen flow. A 200 μL of hexane was added to each tube and briefly vortexed. Tubes were then centrifuged at 18.0 g force (3000 rcf) at 5 °C for 10 min (Eppendorf Centrifuge 5810 R). 50 μL of supernatant was transferred to a glass vial insert for further analysis.
One microliter of the sample was injected by an autosampler (7693A, Agilent Technologies, Palo Alto, USA) to a gas chromatography (GC) system (8890 GC system, Agilent Technologies) coupled with a mass spectrometer www.nature.com/scientificreports/ (MS, 5977B GC/MSD, Agilent Technologies). The gas chromatographic conditions were as followed: a DB-1MS Ultra Inert column (Agilent, 30 m × 0.25 mm I.D. and 0.25 μm film thickness) was used; helium was used as the carrier gas at a constant flow rate of 1 mL/min; a 1 μL volume of sample was injected in a 20:1 split with a split liner (taper, low pressure drop, with glass wool); inlet temperature was at 250 °C; oven temperature was initially held at 150 °C for 1 min, ramped to 276 °C at 21 °°C/min, and held for 15 min with a post-run time of 3 minutes at 325 °C. The MS was run in an SIM mode (selected ion monitoring) for quantitative analysis, using ion fragments for quantitation and verification. The software (ChemStation, Agilent) was used to automatically integrate all peaks and calculate the concentrations of analytes (μg/mL) in the injected hexane solution. These data were exported and the recorded weight of lyophilized feces for each sample was used to calculate concentrations in μg or ng per mg of lyophilized feces. Data for the assessment of bile acids are reported as total amounts in micrograms per milligram of lyophilized fecal content and as percent of total bile acids measured. Total primary bile acids comprise the sum of cholic acid and chenocholic acid, while total secondary bile acids comprise the sum of lithocholic acid, desoxycholic acid, and ursodeoxycholic acid. Total bile acids represent the sum of all bile acids measured by the assay. Furthermore, the ratio of primary to secondary bile acids and the ratio of secondary to primary bile acids were calculated.
Total measured phytosterols comprise the sum of β-sitosterol, brassicasterol, fucosterol, campesterol, sitostanol, and stigmasterol. Total measured zoosterols comprise the sum of cholesterol, coprostanol, cholestenol, and lathosterol. Total measured sterols comprise the sum of phyto-and zoosterols. Furthermore, the ratio of phyto-to zoosterols and the ratio of sterols to fatty acids were calculated. Total measured fatty acids comprise the sum of all fatty acids measured by the assay.

Statistics.
Age and gender between YTE dogs and healthy control dogs were compared by a Wilcoxon rank test. Gender between YTE dogs and healthy Yorkshire Terriers was compared by Fishers exact test. Data were tested for normal distribution by Shapiro Wilk tests. Data were compared between healthy and YTE dogs by use of a Students t-test in case of normal distribution or a Mann Witney U-test in case of non-normal distribution. Data of dogs before and after the treatment trial were compared by a paired T-test or a Wilcoxon signed rank test, dependent on normality testing. Spearman rank test was used to test for correlations between fecal bile acid, sterol, and fatty acid concentrations to clinical, endoscopic, histological scores, the DI, and the fecal abundance of bacterial strains. P < 0.05 were considered significant. Data analysis was performed by SPSS version 20 (SPSS GmbH Software).
Ethical approval. This article does not contain any studies with human participants performed by any of the authors.
Research involving animal rights. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The study was approved by the Ethics Committee of the University of Veterinary Medicine Vienna and the Austrian Federal Ministry of Science and Research (BMWF-68.205/0150-V/3b/2018). The study was carried out with adherence to a high standard (best practice) of veterinary care. Before being enrolled into the study owners of each dog signed a written informed consent form. All methods were performed in accordance with the relevant guidelines and regulations. According to the editorial policy for scientific reports, the study was carried out in compliance with the ARRIVE guidelines.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding
Open Access funding for this article was provided by the University of Veterinary Medicine Vienna (Vetmeduni Vienna).